Andréa Siqueira Haibara

Characterization of a New Selective Antagonist for Angiotensin-(1–7), d-Pro7-Angiotensin-(1–7)

Abstract—Angiotensin-(1–7) [Ang-(1–7)] has biological actions that can often be distinguished from those of angiotensin II (Ang II). Recent studies indicate that the effects of Ang-(1–7) are mediated by specific receptor(s). We now report the partial characterization of a new antagonist selective for Ang-(1–7), d-Pro7-Ang-(1–7). d-Pro7-Ang-(1–7) (50 pmol) inhibited the hypertensive effect induced by microinjection of Ang-(1–7) [4±1 vs 21±2 mm Hg, 25 pmol Ang-(1–7) alone] into the rostral ventrolateral medulla without changing the effect of Ang II (16±2.5 vs 19±2.5 mm Hg after 25 pmol Ang II alone). At 10−7 mol/L concentration, it completely blocked the endothelium-dependent vasorelaxation produced by Ang-(1–7) (10−10 to 10−6 mol/L) in the mouse aorta. The antidiuresis produced by Ang-(1–7) (40 pmol/100 g body weight) in water-loaded rats was also blocked by its analog [1 &mgr;g/100 g body weight; 3.08±0.8 vs 1.27±0.33 mL in Ang-(1–7)–treated rats]. d-Pro7-Ang-(1–7) at a molar ratio of 40:1 did not change the hypotensive effect of bradykinin. Moreover, d-Pro7-Ang-(1–7) did not affect the dipsogenic effect produced by intracerebroventricular administration of Ang II (11.4±1.15 vs 8.8±1.2 mL/h after Ang II) and did not show any demonstrable angiotensin-converting enzyme inhibitory activity in assays with the synthetic substrate Hip-His-Leu and rat plasma as a source of enzyme. Autoradiography studies with 125I–Ang-(1–7) in mouse kidney slices showed that d-Pro7-Ang-(1–7) competed for the binding of Ang-(1–7) to the cortical supramedullary region. In Chinese hamster ovary cells stably transfected with the AT1 receptor subtype, d-Pro7-Ang-(1–7) did not compete for the specific binding of 125I–Ang-II in concentrations up to 10−6 mol/L. There was also no significant displacement of Ang II binding to angiotensin type 2 receptors in membrane preparations of adrenal medulla. These data indicate that d-Pro7-Ang-(1–7) is a selective antagonist for Ang-(1–7), which can be useful to clarify the functional role of this heptapeptide.

Sympathoexcitatory neurotransmission of the chemoreflex in the NTS of awake rats

Cardiovascular responses to chemoreflex activation by potassium cyanide (KCN, 20 microgram/rat iv) were analyzed before and after the blockade of ionotropic or metabotropic receptors into the nucleus of the solitary tract (NTS) of awake rats. Microinjection of ionotropic antagonists [6,7-dinitroquinoxaline-2,3-dione or kynurenic acid (Kyn)] into the lateral commissural NTS (NTSlat), the midline commissural NTS (NTSmid), or into both (NTSlat+mid), produced a significant increase in basal mean arterial pressure, and the pressor response to chemoreflex activation was only partially reduced, whereas microinjection of Kyn into the NTSmid produced no changes in the pressor response to the chemoreflex. The bradycardic response to chemoreflex activation was abolished by microinjection of Kyn into the NTSlat or into NTSlat+mid but not by Kyn microinjection into the NTSmid. Microinjection of alpha-methyl-4-carboxyphenylglycine, a metabotropic receptor antagonist, into the NTSlat or NTSmid produced no changes in baseline mean arterial pressure or heart rate or in the chemoreflex responses. These results indicate that 1) the processing of the parasympathetic component (bradycardia) of the chemoreflex seems to be restricted to the NTSlat and was blocked by ionotropic antagonists and 2) the pressor response of the chemoreflex was only partially reduced by microinjection of ionotropic antagonists and not affected by injection of metabotropic antagonists into the NTSlat or NTSmid or into NTSlat+mid in awake rats.

Autonomic processing of the cardiovascular reflexes in the nucleus tractus solitarii.

The nucleus tractus solitarii (NTS) receives afferent projections from the arterial baroreceptors, carotid chemoreceptors and cardiopulmonary receptors and as a function of this information produces autonomic adjustments in order to maintain arterial blood pressure within a narrow range of variation. The activation of each of these cardiovascular afferents produces a specific autonomic response by the excitation of neuronal projections from the NTS to the ventrolateral areas of the medulla (nucleus ambiguous, caudal and rostal ventrolateral medulla). The neurotransmitters at the NTS level as well as the excitatory amino acid (EAA) receptors involved in the processing of the autonomic responses in the NTS, although extensively studied, remain to be completely elucidated. In the present review we discuss the role of the EAA L-glutamate and its different receptor subtypes in the processing of the cardiovascular reflexes in the NTS. The data presented in this review related to the neurotransmission in the NTS are based on experimental evidence obtained in our laboratory in unanesthetized rats. The two major conclusions of the present review are that a) the excitation of the cardiovagal component by cardiovascular reflex activation (chemo- and Bezold-Jarisch reflexes) or by L-glutamate microinjection into the NTS is mediated by N-methyl-D-aspartate (NMDA) receptors, and b) the sympatho-excitatory component of the chemoreflex and the pressor response to L-glutamate microinjected into the NTS are not affected by the NMDA receptors antagonist, suggesting that the sympatho-excitatory component of these responses is mediated by non-NMDA receptors.

Role of carbon monoxide in l-glutamate-induced cardiovascular responses in nucleus tractus solitarius of conscious rats

Heme oxygenase degrades heme to form carbon monoxide. It has been reported that heme oxygenase-derived carbon monoxide may interact with L-glutamate (L-Glu) receptors in the nucleus tractus solitarius (NTS). Integrative studies suggest that heme oxygenase inhibitors raise blood pressure, in part, by inhibiting carbon monoxide formation in the NTS. The currents studies were designed to determine if heme oxygenase inhibitors affect the cardiovascular actions of L-Glu in the NTS. Accordingly, MAP and HR responses to unilateral microinjections of L-Glu (5 nmol/100 nl) into the NTS were measured before and after ipsilateral microinjections of zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG, 4.5 nmol/100 nl) or chromium mesoporphyrin (CrMP, 1.5 nmol/100 nl) in awake rats chronically instrumented with NTS guide cannulaes and arterial catheters. With respect to non-treatment (+36+/-5 mmHg, -107 bpm, n=10), ZnDPBG pre-treatment attenuated the pressor and bradycardic responses to L-Glu (+7+/-3 mmHg, -10+/-6 bpm, P<0.05). CrMP similarly attenuated cardiovascular responses to L-Glu (+47+/-3 mmHg, -68+/-8 bpm vs. +20+/-5 mmHg, -40+/-9 bpm; before vs. after, n=10, P<0.05). Matched series yielded no vehicle- or time-related effects. Our findings suggest that a heme oxygenase product, such as carbon monoxide, may affect NTS glutamatergic neurotransmission to participate in cardiovascular control.

Effects of low-protein diet on the baroreflex and Bezold-Jarisch reflex in conscious rats.

The present study evaluated the effects of a low-protein diet (LP, 6% protein) on cardiovascular reflexes of Male Fisher rats. Three experimental groups, and their respective controls (15% protein), were used. (1) Baroreceptor reflex (BAR); (2) Bezold-Jarisch reflex (BJR); and (3) Prazosin treated. Dietary restriction began after weaning (three weeks) and lasted for a period of five weeks, after which animals were subjected to the experimental protocols. The BAR group was evaluated through injections of phenylephrine (0.55.0 μg/Kg, i.v.) and sodium nitroprusside (0.77.0 μg/Kg, i.v.) while the BJR was evaluated through injections of serotonin (2.510μg/Kg, i.v.). Our results showed an increased baroreflex gain bradycardia for the LP group (-0.96 ± 0.34 vs. -2.12 ± 1.06 bpm/mmHg) and a larger bradycardia for the BJR the LP group (160 ± 18% greater than controls). Basal cardiovascular parameters were not different between LP and control rats, however LP animals treated with prazosin resulted in a larger fall of blood pressure (-19±3 vs. -28±5 mmHg). In conclusion, LP rats present an increased responsiveness of BAR and BJR, which could contribute to their normal levels of cardiovascular parameters, in spite of the possible increase in the sympathetic vasomotor tonus observed with the prazosin protocol.

Altered cardiovascular reflexes responses in conscious Angiotensin-(1-7) receptor Mas-knockout mice.

This study evaluated the physiological importance of Angiotensin-(1-7) receptor Mas on reflex control of circulation. Experiments were performed in male Mas-knockout (Mas-KO) and Wild Type (WT) conscious mice (12-20 wk of age). Baroreceptor reflex was evaluated by the bradycardic response induced by phenylephrine (0.25 μg/5 μl, i.v.). Bezold-Jarisch reflex was evaluated by phenylbiguanide (0.5 μg/5 μl, i.v.) and chemoreflex by potassium cyanide (2.5 μg/5 μl, i.v.). Baseline mean arterial pressure was higher in Mas-KO (n=14) as compared with WT mice (n=18) (118±1 mmHg vs. 109±2 mmHg); however, heart rate was similar in both strains (615±30 bpm vs. 648±13 bpm). Baroreflex bradycardia was lower (0.78±0.44 ms/mmHg vs. 1.30±0.14 ms/mmHg) in Mas-KO compared with WT mice. The depressor (-17±5 mmHg vs. -45±6 mmHg) and bradycardic (-212±36 bpm vs. -391±29 bpm) components of the Bezold-Jarisch reflex were also lower in Mas-KO mice. In addition, chemoreflex pressor response (+20±3 mmHg vs. +12±0.8 mmHg) and bradycardic response (-250±74 bpm vs. -52±26 bpm) were significantly higher in Mas-KO. These results further advances previous studies by showing that the lack of Mas receptor induced important imbalance in the neural control of blood pressure, altering not only the baroreflex but also the chemo- and Bezold-Jarisch reflexes.

Involvement of the parabrachial nucleus in the pressor response to chemoreflex activation in awake rats

Activation of the chemoreflex with potassium cyanide (KCN, 40 microg/rat, i.v.) in awake rats produces pressor and bradycardic responses as well as a tachypneic response. In the present study, we evaluated the involvement of the periaqueductal gray matter (PAG) and the parabrachial nucleus (PBN) in the neural pathways of the cardiovascular responses to chemoreflex activation. The cardiovascular responses to chemoreflex activation were evaluated before and after bilateral microinjection of 2% lidocaine, a local anesthetic, into the PBN or PAG in order to block in a reversible manner the neuronal activity and axonal conduction of fibers of passage in these areas. The data show that the pressor response to chemoreflex activation 3 min after bilateral microinjection of lidocaine into the dorsolateral aspect of the PBN was significantly reduced in comparison to the control response (32 +/- 5 vs. 48 +/- 4 mm Hg, n = 7), with no significant changes in the bradycardic responses. The effect of lidocaine was reversible since the pressor response was back to control levels 15 min after microinjection of this anesthetic. Bilateral microinjections of lidocaine into the dorsolateral (n = 11) or lateral (n = 8) columns of the PAG in distinct groups of rats produced no significant changes in the pressor or bradycardic responses of the chemoreflex. These data indicate that the PBN is part of the neuronal pathways involved in the sympathoexcitatory component of the chemoreflex while the PAG is not.

Physical Exercise Performance in Temperate and Warm Environments Is Decreased by an Impaired Arterial Baroreflex

The present study aimed to investigate whether running performance in different environments is dependent on intact arterial baroreceptor reflexes. We also assessed the exercise-induced cardiovascular and thermoregulatory responses in animals lacking arterial baroafferent signals. To accomplish these goals, male Wistar rats were subjected to sinoaortic denervation (SAD) or sham surgery (SHAM) and had a catheter implanted into the ascending aorta to record arterial pressure and a telemetry sensor implanted in the abdominal cavity to record core temperature. After recovering from these surgeries, the animals were subjected to constant- or incremental-speed exercises performed until the voluntary interruption of effort under temperate (25° C) and warm (35° C) conditions. During the constant-speed exercises, the running time until the rats were fatigued was shorter in SAD rats in both environments. Although the core temperature was not significantly different between the groups, tail skin temperature was higher in SAD rats under temperate conditions. The denervated rats also displayed exaggerated increases in blood pressure and double product compared with the SHAM rats; in particular, in the warm environment, these exaggerated cardiovascular responses in the SAD rats persisted until they were fatigued. These SAD-mediated changes occurred in parallel with increased variability in the very low and low components of the systolic arterial pressure power spectrum. The running performance was also affected by SAD during the incremental-speed exercises, with the maximal speed attained being decreased by approximately 20% in both environments. Furthermore, at the maximal power output tolerated during the incremental exercises, the mean arterial pressure, heart rate and double product were exaggerated in the SAD relative to SHAM rats. In conclusion, the chronic absence of the arterial baroafferents accelerates exercise fatigue in temperate and warm environments. Our findings also suggest that an augmented cardiovascular strain accounted for the early interruption of exercise in the SAD rats.

Sinoaortic denervation prevents enhanced heat loss induced by central cholinergic stimulation during physical exercise

The present study investigated whether the effects of central cholinergic stimulation on thermoregulation during exercise are modulated by arterial baroreceptors. Wistar rats were submitted to sinoaortic denervation (SAD) or sham denervation (SHAM) and then fitted with a chronic guide cannula into the lateral cerebral ventricle. After 2 weeks, a catheter was implanted into the ascending aorta, and a temperature sensor was implanted into the peritoneal cavity. Two days later, the rats were submitted to exercise on a treadmill at 18 m/min until fatigued. Thermoregulatory and cardiovascular responses were measured after injection of 2 μL of 10mM physostigmine (Phy) or 0.15M NaCl solution (Sal) into the cerebral ventricle. In SHAM rats, Phy injection induced a greater exercise-induced increase in blood pressure and lower increase in heart rate than Sal treatment. In the SAD group, the attenuation of heart rate in response to Phy was blocked despite an exaggerated increase in blood pressure. SHAM rats treated with Phy had a higher increase in tail skin temperature compared to Sal injection (31.9 ± 0.4 °C Phy-SHAM vs. 30.1 ± 0.6 °C Sal-SHAM, 5 min after injection; p<0.05), resulting in a lower exercise-induced increase in core temperature. In contrast, SAD blocked the Phy injection effects in thermoregulatory responses during exercise (tail temperature: 30.1 ± 1.2 °C Phy-SAD vs. 29.5 ± 1.2 °C Sal-SAD, 5 min, p = 0.65). Therefore, we conclude that the enhancement of cutaneous heat loss induced by central cholinergic stimulation during exercise is mediated primarily by arterial baroreceptors.

The hypothalamic paraventricular nucleus and carotid receptors modulate hyperglycemia induced by hemorrhage

The aim of this study was to assess the role of cholinergic transmission in the paraventricular nucleus of the hypothalamus (PVN) and carotid body receptors in mediating a rise in plasma glucose levels in response to hemorrhagic hypotension in rats. Methylatropine (1x10(-9) mol) or 0.15 M NaCl (0.2 microl) was injected into the PVN of Wistar rats weighing 250-300 g bearing a chronic jugular catheter for blood sampling and hemorrhage (1.2 ml/100 g/2 min). Polyethylene cannulae (PE-10) were inserted into the left femoral artery for cardiovascular monitoring. In the other experimental protocol, hemorrhage was performed on rats submitted to bilateral carotid receptor denervation (H-CD). The results show that the hyperglycemic response to hemorrhage was decreased by either methylatropine (H-MA) treatment or bilateral carotid receptor denervation (10.3+/-0.4 mM, control, n=15 vs. 7.7+/-0.2 mM, H-MA, n=12, and 7.6+/-0.3 mM, H-CD, n=5, p<0.01). Furthermore, methylatropine did not affect the recovery of blood pressure after hemorrhage-induced hypotension, suggesting that the metabolic and pressor adjustments have different efferent pathways. Our data demonstrate that cholinergic input from the PVN and carotid receptors (chemo- and/or baroreceptors) might participate in the same neural pathway activated by hemorrhage-induced hypotension that produces hyperglycemia.